“SARS-COV-2” TEM Images: Game Over

They say that “a picture is worth a thousand words” and that “seeing is believing.” When it comes to “viruses,” these pictures come in the form of Transmission Electron Microscope (TEM) imaging. These are the scary depictions of the brightly colored and digitally enhanced particles you see plastered all over the media selling you on the idea that a novel “virus” known as “SARS-COV-2” actually exists.

The representative “corona” particle has been shared over and over again, making its way into t-shirts, posters, toys, video games, and more.

As the best symbols often do, the “coronavirus” has embedded itself deep into the collective consciousness. Most do not question whether or not the TEM images these symbols are based upon are legit nor how they were actually aquired. People believe wholeheartedly in the authenticity of these depictions of the spiked particle that has been repeatedly advertised to them in various forms over the last few years.

However, there is another quote by the popular poet Edgar Allen Poe which says to “believe only half of what you see and nothing of what you hear.” When it comes to TEM images of the particles claimed to be “viruses,” there are very good reasons to put them in the category of the things you see that you should definitely not believe in. For starters, the work of Dr. Harold Hillman, MB, BSc, MRCS, PhD showed that most of the structures and particles seen in electron microscope imagery are in fact artefacts created through the dehydration, embedding, fixing, and staining processes the samples go through before being imaged. As nothing can survive these processes, anything within the sample is in a dead and unnaturally altered state. I have provided a few quotes from Dr. Hillman himself explaining the problems with TEM imaging:

‘What Price Intellectual Honesty?’ asks a neurobiologist

“I was so disturbed by the thought that subcellular fractionation might be an unsatisfactory technique that I decided to take a completely different technique and subject it to a smaller analysis. I took electron microscopy, asking the question, ‘How much does a picture taken with this instrument tell one about the structure of the living cell?’ Since the early 1950s, there has been a passion for relating ‘structure’ to ‘function’, that is, the appearance by electron microscopy of a particular identifiable part of a cell with the biochemistry it exhibits.

The light microscope had been used to examine living cells, unfixed tissue and stained sections, for 100 years until the 1940’s. At that time, the electron microscope was introduced. It permits much higher resolution and magnification than the light microscope, but the tissue can not survive the low pressure, the bombardment of electrons and x-radiation in the electron microscope, so it has to be coated with a deposit of salts of osmium lead or tungsten, which is not destroyed by these agents, and can therefore be examined. Cytologists were very anxious to use this more powerful instrument to look at the fine structure of cells.”

“For example, most cytologists know, but readers of elementary textbooks do not, that when one looks at an illustration of an electron micrograph: an animal has been killed; it cools down; its tissue is excised; the tissue is fixed (killed); it is stained with a heavy metal salt; it is dehydrated with increasing concentrations of alcohol; it shrinks; the alcohol is extracted with a fat solvent, propylene oxide; the latter is replaced by an epoxy resin; it hardens in a few days; sections one tenth of a millimetre thick, or less, are cut; they are placed in the electron microscope, nearly all the air of which is pumped out; a beam of electrons at 10,000 volts to 3,000,000 volts is directed at it; some electrons strike a phosphorescent screen; the electron microscopists select the field and the magnification which show the features they wish to demonstrate; the image may be enhanced; photographs are taken; some are selected as evidence. One can immediately see how far the tissue has travelled from life to an illustration in a book.”

“I have shown, to my own satisfaction that (i) at least some popular important biochemical research techniques have never been controlled, (ii) most of the new structures in cells apparent by electron microscopy are artifacts, (iii) there are only nerve cells and naked nuclei in a ground substance in the brain and spinal cord, (iv) there are no synapses, (v) the transmitter hypothesis is doubtful. I have published all the evidence for these statements, although this has not always been easy.”

https://www.big-lies.org/harold-hillman-biology/what-price-intellectual-honesty.htm

Dr. Hillman produced and published evidence showing that the processes used to create any TEM image significantly changes the properties of the tissues or samples being subjected to them. He showed that the structures and particles seen are artefacts and that the studies producing these images lack the necessary and proper controls which would prove his assertions to be correct. The above brief paragraphs from Dr. Hillman do not do his work justice as the evidence he uncovered went a long way towards challenging the accepted scientific dogma that TEM images are valid proof of anything at all. Even if one were to ignore the work of Dr. Hillman and still believe that the particles picked out from a sea of billions of similar and identical ones actually depict “viruses,” there is another very striking reason to re-examine that position.

Identity Crisis

As the “coronavirus” craze went into full swing in early 2020, numerous studies popped up purporting to show TEM images of “SARS-COV-2” particles in different tissues and organs outside of the lungs. These studies were used to justify claims that the “virus” was attacking various organs such as the kidneys. Virologists and mainstream media propaganda claimed that these images were proof of the “virus” and that “SARS-COV-2” could be shown to spread throughout the body. However, other researchers started to question the validity of the images from these studies, claiming that the particles depicted were not “SARS-COV-2,” but were in fact other subcellular vesicles. These researchers knew what most people do not, which is that the particles said to be “viruses” can not be distinguished from normal “non-viral” subcellular particles like ubiquitous coated vesicles, such as clathrin-coated vesicles, or COPI- or COPII-coated vesicles, as well as multivesicular bodies (MVB’s) and exosomes. Even the crown-like coating supposedly specific to the CORONA (a.k.a. crown) “virus” can be seen on these “non-viral” particles. Thus many of the TEM images of “SARS-COV-2” were rightfully challenged.

In July 2020, a group of researchers weighed in on this debate and carried out their own experiments to see whether or not the particles said to be “SARS-COV-2” could be picked out from amongst the many similar and identical ones in the TEM images. To do so, they took samples from the lungs and kidneys of four “Covid-19” patients. They used RT-PCR to determine that there was “viral” RNA within the lungs but not within the kidneys. Upon ultrastructural examination, the researchers found “coronavirus-like” particles within the lung samples with “viral” RNA. However, they also found identical ones in the kidney samples without “viral” RNA. The researchers even found the exact same “corona-like” particles in samples from 2 patients without “SARS-COV-2” which were taken during and before the “pandemic.” They even performed semiquantitative analysis of these intracellular structures in different types of renal cells from 20 preimplantation donor kidney biopsies, before and during the outbreak of “SARS-CoV-2,” with negative RT-PCR for “SARS-CoV-2” RNA in order to demonstrate that these cell structures are numerous and ubiquitous in various types of cells. The researchers concluded that TEM alone was not valid proof and that these images could not be relied upon in order to claim “viral” invasion:

SARS-CoV-2 Virions or Ubiquitous Cell Structures? Actual Dilemma in COVID-19 Era

“Several reports have suggested ultrastructural evidence of direct infection of different types of kidney cells by severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) in postmortem analysis and kidney biopsy specimens in patients with proven viral reverse-transcriptase polymerase chain reaction (RT-PCR) from nasopharyngeal smears. Detection of supposed viral particles by transmission electron microscopy (TEM) was used as sufficient evidence for viral invasion of renal tissue but data regarding detection of viral RNA or other valuable methods for viral detection of kidney specimens were missing. Likewise, an additional study proposed direct SARS-CoV-2 infection of endothelial cells in multiple organs of patients with coronavirus disease 2019 (COVID-19) on the basis of TEM solely. The presented particles in the aforementioned studies exhibit a diameter of 50 to 150 nm and crown-like electron-dense coat, so they may appear similar to Coronavirus but also similar to ubiquitous coated vesicles, such as clathrin-coated vesicles, or COPI- or COPII-coated vesicles. Moreover, clusters of viral particles inside the vacuole might resemble multivesicular bodies (MVBs), which are regular structures of the endocytic pathway.

Herein, to detect direct invasion of SARS-CoV-2 in the kidney, we performed RT-PCR on fresh postmortem lung and kidney specimens of 4 patients with COVID-19. In all 4 patients, viral RNA was confirmed in all lung samples, but was negative in all kidney samples. However, ultrastructural examination revealed intracellular vesicular structures of similar size and morphology in lung with proven viral RNA and in kidney with no viral RNA. In lung specimens with proven viral RNA, we observed many structures that could be either viral particles with typical corona or coated vesicles with electron-dense protein coat (Figure 1a). In addition, in the same SARS-CoV-2–positive lung specimens, we found vacuoles that could be either membrane-bound clusters of viral particles or MVBs with intralumenal vesicles inside (Figure 1c). On the other hand, ultrastructural examination of the lung specimens of 2 patients without SARS-CoV-2 (1 autopsy specimen of lung with negative RT-PCR for viral RNA and 1 biopsy specimen of lung before COVID era) revealed the same structures, resembling viral particles, coated vesicles, or MVBs, as in a specimen with positive SARS-CoV-2 (Figure 1b and d).

TEM analysis of postmortem kidney specimens of patients with COVID-19 revealed numerous individual vesicles and clusters of vesicles in different types of kidney cells, despite negative RT-PCR for SARS-CoV-2 RNA (Figure 1e and f). We additionally performed semiquantitative analysis of these intracellular structures in different types of renal cells from 20 preimplantation donor kidney biopsies, before and during the outbreak of SARS-CoV-2, with negative RT-PCR for SARS-CoV-2 RNA to demonstrate that these cell structures are numerous and ubiquitous in various types of cells (Table 1).

There has been increasing evidence to indicate that coated vesicles and MVBs may mimic viral particles. However, it is known that the budding of enveloped viruses (to which SARS-CoV-2 belongs) from the plasma membrane, or the limiting membrane of the endosome, resembles the formation of intralumenal vesicles inside MVBs. Moreover, the 2 processes share some components of the same protein machinery. Indeed, we detected intralumenal vesicles budding from discontinued limiting membrane of the vacuole in lung endothelial cells with positive SARS-CoV-2 RNA (Figure 1a). This finding strongly suggests that this structure is not MVBs but rather a cluster of viral particles with some of budding virions. Indisputable evidence of virions would thus be provided only by immunoelectron microscopy. In addition, coated vesicles might also faintly resemble viral particles, but it is necessary to be cautious about the intracellular location of coated vesicles. Specifically, coated vesicles are transient and are therefore mostly found in close vicinity to the membrane from which they bud, because they shed their coat within seconds after their formation.

To conclude, although TEM may serve as a useful diagnostic method for the detection of viral infection, caution should be exercised when confirmation of viral invasion relies only on TEM. Additional convincing methods, including immunoelectron microscopy, immunohistochemistry, and viral genetic material analysis, are needed for indisputable proof of viral invasion in organs.”

https://www.kireports.org/article/S2468-0249(20)31368-1/fulltext

That the particles claimed as “SARS-COV-2” were found not only in samples without “viral” RNA but also in samples from patients without “SARS-COV-2” from the pre-“Covid-19” era should be enough for anyone being intellectually honest to conclude that the particles in the TEM images are nothing but a meaningless representation. It is admitted that the same “SARS-COV-2” particles can be microvesicular bodies, clathrin-coated vesicles, golgi apparatus, secretory granules and vesicles, coatomer-coated vesicles, and/or exosomes. While the images can be anything to the eye of the beholder, what they can not be is valid proof of “viruses.”

A little over a year later in September 2021, the same researchers published another study on the subject of misinterpreting TEM images. This time, while they still acknowledged that using TEM to identify “viruses” can be misleading and alone is not suitable as proof, they aimed to correct this problem using their own method which was a variant of correlative microscopy that combined immunohistochemical labelling with TEM. Essentially, the researchers used theoretical antibodies in an attempt to prove theoretical “viruses” by claiming that the antibodies targeted and labelled the particles that were “SARS-COV-2.” In this approach, the researchers claim that they could distinguish the particles that are “SARS-COV-2” from those that are not “SARS-COV-2″…even though the particles are identical in every other way.

There are many issues with this theory and ultimately their evidence. First of all, as antibodies have never been properly purified nor isolated themselves, one can not use one fictional creation to validate another fictional creation. Even if antibodies had been scientifically proven to exist, without having purified and isolated the particles claimed to be “SARS-COV-2,” there can be no way to know which theoretical antibodies would be specific to the “virus” itself. Antibodies have been repeatedly shown to be non-specific and can bind to many other proteins other than the intended target. Studies relying on antibodies are rarely reproduced which has led to reproducibility crisis resulting in false and erroneous results making up the majority of scientific research.

The researchers also provided many other reasons within their own paper as to why the methods they used as “proof” are unreliable. It was stated that:

1. Immunohistochemistry can be falsely positive or negative due to several reasons in the pre-analytical or analytical phase.
2. Antigens are known to be vulnerable at several stages of the immunohistochemical procedure and also during specimen preparation for TEM.
3. Conventional TEM analysis strongly depends on tissue preservation, which might distort the structure of “viruses” if it is not performed optimally.
4. The sample used for TEM analysis might miss the area containing “viruses.”
5. An IHC reaction at a light microscopy level could be misleading due to misinterpretation of nonspecific staining.
6. Low magnification and resolution of IHC do not allow pathologists to see “virions” or be convinced that an IHC reaction colocalizes with “virions.”
7. Despite the possibility to see the structures (“virions”) with TEM due to its high resolution, they can be misinterpreted.
8. In order to get a “specific” and “reliable” immunoreaction, it crucially depends on the immunolabelling protocol, from the first steps of tissue sample preparation to the choice of antibody.

The paper itself goes into great detail as to what is actually done to prepare the samples for both immunohistochemistry and TEM imaging. I highlighted these sections to showcase the numerous processes and substances to which the sample undergoes for imaging. As Dr. Hillman pointed out, the images of the end result after the multiple alterations done to the sample can hardly be considered an accurate representation of what was taken from within a living being.

Ultimately, the researchers discovered that appropriate fixation and embedding mediums are crucial to getting the results that they desired as this can restrict both antibody access and antibody quality. The results of the immunogold labelling of “SARS-CoV-2” strongly depended on the selection of the primary antibody and resin as they were only able to get a “specific” staining and a satisfactory ultrastructure on Lowicryl sections with an “appropriate” antibody. The researchers tried various other methods utilizing other “SARS-COV-2 specific” antibodies with ultrathin Epon sections which all failed to produce the results that they wanted to see. Oddly enough, unlike their previous report which actually had a control of negative “SARS-COV-2” patients which showed the same particles are found in those without the disease, the researchers did not attempt to see if the same “specific” immunolabelling results could be obtained using samples from “SARS-COV-2” negative individuals.

Presented below is the full paper for what they claim as indisputable proof that “SARS-COV-2” was found in infected tissues. Based on the laundry list of problems mentioned above, this was obviously not the case:

Just Seeing Is Not Enough for Believing: Immunolabelling as
Indisputable Proof of SARS-CoV-2 Virions in Infected Tissue

“There is increasing evidence that identification of SARS-CoV-2 virions by transmission electron microscopy could be misleading due to the similar morphology of virions and ubiquitous cell structures. This study thus aimed to establish methods for indisputable proof of the presence of SARS-CoV-2 virions in the observed tissue. Methods: We developed a variant of the correlative microscopy approach for SARS-CoV-2 protein identification using immunohistochemical labelling of SARS-CoV-2 proteins on light and electron microscopy levels. We also performed immunogold labelling of SARS-CoV-2 virions. Results: Immunohistochemistry (IHC) of SARS-CoV-2 nucleocapsid proteins and subsequent correlative microscopy undoubtedly proved the presence of
SARS-CoV-2 virions in the analysed human nasopharyngeal tissue. The presence of SARS-CoV-2 virions was also confirmed by immunogold labelling for the first time. Conclusions: Immunoelectron
microscopy is the most reliable method for distinguishing intracellular viral particles from normal cell structures of similar morphology and size as virions. Furthermore, we developed a variant of correlative microscopy that allows pathologists to check the results of IHC performed first on routinely used paraffin-embedded samples, followed by semithin, and finally by ultrathin sections. Both methodological approaches indisputably proved the presence of SARS-CoV-2 virions in cells.

1. Introduction

Since SARS-CoV-2 emerged, many studies have reported alleged SARS-CoV-2 virions in COVID-19 patients’ tissues found by transmission electron microscopy (TEM) [1–3]. Detection of viral particles by TEM only was used as sufficient evidence for viral identification in the aforementioned studies, indicating evidence of direct viral infection of certain cells and tissues. However, few authors have drawn attention to the possible misinterpretation of these findings due to the similarity of structure and size between virions and normal cell structures [4,5]. In a previous study, we showed cell structures resembling SARS-CoV-2 virions in lung and kidney specimens of SARS-CoV-2 positive and negative patients based on their RT-PCR results for SARS-CoV-2 RNA [6]. In light of our results, we therefore suggested that for unambiguous identification of virions in cells and tissues, methods such as immunohistochemistry or immunoelectron microscopy are needed, in addition to viral genetic material analysis.

Existing methods used for viral detection in tissue specimens have known limitations. For example, immunohistochemistry can be falsely positive or negative due to several reasons in the pre-analytical or analytical phase. Furthermore, conventional TEM analysis strongly depends on tissue preservation, which might distort the structure of viruses if it is not performed optimally. An additional disadvantage of TEM analysis is that the sample might miss the area containing viruses [7]. On the other hand, immunoelectron microscopy is a very powerful and highly sophisticated method for the detection and identification of viruses, but is mostly available in academic settings. The method that connects the aforementioned methods and also represents a bridge between light and
electron microscopy is correlative microscopy.

We performed a variant of correlative immunohistochemistry and immunogold labelling of SARS-CoV-2 proteins for indisputable proof of the presence of SARS-CoV-2 virions in nasopharyngeal tissue of patients with positive RT-PCR for SARS-CoV-2 RNA. We combined immunohistochemical labelling of SARS-CoV-2 proteins on light and electron microscopy levels and thus developed a correlative microscopy approach for SARS-CoV-2
protein identification, for which there are no reports in the literature to date. We also showed that a specific and reliable immunoreaction crucially depends on the immunolabelling protocol, from the first steps of tissue sample preparation to the choice of antibody. We believe that immunolabelling, if performed correctly, is the most reliable method of distinguishing intracellular viral particles from normal cell structures of similar structure and size to virions. To the best of our knowledge, this is the first report of the immunolabelling of SARS-CoV-2 virions on an electron microscopy level.

2. Materials and Methods

2.1. Patients

Autopsy specimens from nasopharyngeal tissue of SARS-CoV-2 positive patients by RT-PCR were taken for immunohistochemistry and immunogold labelling of SARS-CoV-2 virions.

2.2. Immunohistochemistry (IHC) for Correlative and Transmission Electron Microscopy (TEM)

Nasopharyngeal tissue pieces (5 mm3) were fixed in 4% paraformaldehyde (Merck, Darmstadt, Germany) for 24 h, dehydrated in ethanol, and embedded in paraffin. The tissue sample for immunohistochemistry was obtained by punching the paraffin block with a manual tissue punch. Punched paraffin blocks were then deparaffinized and rehydrated with ethanol. Antigen retrieval was performed with microwave heating in 10 mM sodium citrate buffer, pH 6.0 (Sigma Aldrich, Burlington, MA, USA) for 20 min. Endogenous peroxidases were blocked by 3% hydrogen peroxide (Ventana, Monterey, CA, USA) for 15 min following incubation in primary rabbit antibodies against SARS-CoV-2 nucleocapsid protein (1:600; NB100, Novus Biologicals, Cambridge, UK) for 2 h at room temperature. After washing with reaction buffer (Ventana, Monterey, CA, USA), an UltraView Universal DAB Detection Kit (Ventana, Monterey, CA, USA) was used with the following protocol: HRP enzyme conjugated to goat anti-rabbit secondary antibodies for 20 min, reaction buffer (Ventana, Monterey, CA, USA) twice for 5 min, a mixture of 3,3’-diaminobenzidine
(DAB) chromogen and H2O2 for 20 min, and finally washed with reaction buffer (Ventana, Monterey, CA, USA) and distilled water for 5 min.

After IHC, tissue samples were transferred to 0.1 M Millonig’s phosphate buffer overnight and processed the next day for transmission electron microscopy. They were first post-fixed in 1% OsO4 (Merck, Darmstadt, Germany) for 30 min, then dehydrated in graded concentrations of ethanol and propylene oxide 1-2-propylene oxide (Merck, Darmstadt, Germany) for 10 min in each solution. Tissue pieces were then incubated in a mixture (1:1) of 1-2-propylene oxide and Epon 812 resin (Serva Electrophoresis, Heidelberg, Germany) for 20 min, embedded in 100% Epon, and polymerized at 60 ◦C for 24 h. The next day, semithin sections of 1 µm were cut with an ultramicrotome Leica EM UC6 and stained with Azure II staining solution to find SARS-CoV-2-positive cells as the region of interest, to select this region for ultrathin sectioning. Ultrathin sections of 60 nm were cut and viewed in a transmission electron microscope JEM- 1200 EXII (JEOL, Tokyo, Japan) at 60 kV (Figure 1).

2.3. Immunogold Labelling on Ultrathin Sections

For immunogold labelling on ultrathin Epon sections, small samples (2 mm3) of nasopharyngeal tissue were fixed in a mixture of 4% paraformaldehyde (Merck, Darmstadt, Germany) and 2% glutaraldehyde (Serva, Heidelberg, Germany) in 0.2 M cacodylate buffer (pH 7.3) for 3 h at 4 ◦C. Overnight rinsing in 0.33 M sucrose in 0.2 M cacodylate buffer was followed by post-fixation with 1% OsO4 (Serva, Heidelberg, Germany) for 1 h. After
dehydration in an ethanol series, tissue samples were embedded in Epon 812 resin (Serva Electrophoresis, Heidelberg, Germany) and cut into ultrathin sections (60 nm thick), which were collected on nickel grids. Antigen unmasking treatment was performed by incubation of some grids in a humid chamber on a large drop of a saturated aqueous solution of sodium meta periodate (Merck, Darmstadt, Germany) for 1 h at room temperature. Grids were then washed in distilled water. Non-specific labelling was blocked by blocking buffer (5% foetal calf serum in 0.1% BSA in PBS (washing buffer)) for 30 min at room temperature. Primary antibodies against SARS-CoV-2 nucleocapsid protein (NB100; Novus Biologicals, Cambridge, UK) diluted 1:50 or SARS-CoV-2 spike glycoprotein S1 (ab 275759; Abcam, Cambridge, UK) diluted 1:100 was applied and incubated overnight at 4 ◦C. After washing in washing buffer, goat anti-rabbit secondary antibodies with 18 nm gold (Au) diluted 1:40 in blocking buffer were applied for 90 min. In all cases, the manufacturers of the antibodies used provided proof of validation of the technical specifications. In all cases, negative controls were also performed, in which primary antibodies were replaced with PBS. Sections were counterstained with uranyl acetate and lead citrate.

For immunogold labelling on ultrathin Lowicryl sections, small cubes (1 mm 3) of nasopharyngeal tissue were transferred to 2% paraformaldehyde (Merck, Darmstadt Germany) plus 0.05% glutaraldehyde (Serva, Heidelberg, Germany) in PBS for 1 h at room temperature. Samples were dehydrated by progressively lowering the temperature, then
embedded in Lowicryl HM20 resin (Polysciences, Eppelheim, Germany) in a Leica AFS apparatus (Leica Microsystems, Vienna, Austria), according to the following protocol: 30% ethanol for 30 min at 0 ◦C, 55% ethanol for 30 min at −15 ◦C, 70% ethanol for 30 min at −30 ◦C, 100% ethanol for 1 h at −50 ◦C, 75% ethanol/25% HM20 for 1 h at −50 ◦C, 50% ethanol/50% HM20 for 1 h at −50 ◦C, 25% ethanol/75% HM20 for 1 h at −50 ◦C, 100% HM20 for 1 h, and 100% HM20 overnight at −50 ◦C. HM20 was polymerized for 48 h at −50 ◦C and then for 24 h at −20 ◦C under UV light. Ultrathin sections (60 nm thick) were cut and collected on nickel grids. Sections were washed in washing buffer (0.1% Na-azide, 0.8% BSA, and 0.1% IGSS gelatine in PBS), blocked in blocking buffer (5% foetal calf serum in 0.1% BSA in PBS (washing buffer)) for 30 min at room temperature, and incubated overnight at 4 ◦C with primary antibodies against SARS-CoV-2 nucleocapsid protein (NB100-56576; Novus Biologicals, Cambridge, UK) diluted 1:50 or SARS-CoV-2 spike glycoprotein S1 (ab 275759; Abcam, Cambridge, UK) diluted 1:100. After washing in washing buffer, goat anti-rabbit secondary antibodies with 18 nm gold (Au) diluted 1:40 in blocking buffer were applied for 90 min. Sections were counterstained with uranyl acetate and lead citrate. Ultrathin sections were observed in a Philips CM100 transmission electron microscope (Philips, Amsterdam, The Netherlands) at 80 kV (Figure 1).

3. Results

3.1. Correlative Microscopy Approach and SARS-CoV-2 Virion Detection

The results of immunohistochemistry of SARS-CoV-2 nucleocapsid proteins and subsequent correlative microscopy undoubtedly proved the presence of SARS-CoV-2 virions in the analysed human nasopharyngeal tissue (Figure 2). We detected cells with positive
immunohistochemical staining in semithin sections by light microscopy (Figure 2a). The same cells with positive immunohistochemical staining were examined on ultrathin Epon sections, showing electron-dense material in the cytoplasm inside endosomes (Figure 2b,c). Finally, at higher magnification, we identified in endosomes of the observed cell virions marked with a fine granular electron-dense reaction corresponding to the product of DAB (Figure 2d).

3.2. Immunoelectron Microscopy Approach and SARS-CoV-2 Virion Detection

Through immunogold labelling, we confirmed the presence of SARS-CoV-2 virions in human nasopharyngeal tissue samples. The results of this method greatly depended on the type of ultrathin sections and primary antibodies used in the immunolabelling protocol (Figure 3). Immunogold reaction on ultrathin Epon sections with primary antibodies against SARS-CoV-2 nucleocapsid protein (Novus Biologicals) was ubiquitous and therefore unspecific (Figure 3a), while on ultrathin Epon sections with primary antibodies against SARS-CoV-2 spike glycoprotein S1 (Abcam), it was scarce and unspecific (Figure 3b). After an antigen unmasking procedure on ultrathin Epon sections (described in Methods) with primary antibodies against SARS-CoV-2 spike glycoprotein S1 (Abcam), immunogold labelling was more specific but weak (Figure 3c). Immunogold labelling on ultrathin Lowicryl sections with primary antibodies against SARS-CoV-2 spike glycoprotein S1 (Abcam) was specific and intense (Figure 3d).

4. Discussion

Viral genetic material analysis of patients’ nasopharyngeal tissue by itself is not sufficient proof of SARS-CoV-2 active infection in body tissues, but a positive RT-PCR test, which is currently an established test for SARS-CoV-2 infection, is a precondition for further analysis of SARS-CoV-2 virus presence in a particular tissue. Likewise, TEM analysis per se is not enough for unequivocal proof of SARS-CoV-2 virions in the tissue of interest due to the similar structure and size of these virions and ubiquitous cell structures [4,5]. Thus, we emphasised in a previous study that after a positive RT-PCR test for SARS-CoV-2 RNA, IHC or even immunoelectron microscopy is needed for the reliable identification of virions in suspected infected tissues.

In this study, we present two approaches to immunolabelling on autopsy nasopharyngeal tissue samples of patients with positive RT-PCR for SARS-CoV-2 RNA. We thus performed correlative microscopy in which we combined the advantages of both IHC and electron microscopy to analyse the same specimen. We first performed an immunohistochemical reaction against SARS-CoV-2 nucleocapsid protein with diaminobenzidine (DAB) chromogen on paraffin sections, which is a standard diagnostic method in our pathology laboratory. DAB is oxidised in the presence of peroxidase and hydrogen peroxide, then bound on secondary antibodies, resulting in the deposition of a brown-coloured precipitate at the site of enzymatic activity, visible under light microscopy and also by electron microscopy, especially after post-fixation with osmium [8]. We also performed the same immunohistochemical protocol on deparaffinized small tissue pieces that were then fixed in osmium, embedded in Epon resin, and cut into semithin sections. Finally, the selected area of cells with positive immunohistochemical staining was cut for TEM. The immunohistochemical reaction enabled visualisation of immunohistochemically labelled SARS-CoV-2 virions in infected cells on a semithin section by light microscopy, followed by TEM detection of SARS-CoV-2 virions in the same cell on ultrathin sections. We managed to preserve immunohistochemical labelling of virions with DAB during the TEM embedding procedure and showed that IHC and TEM can be used as analytical methods for unambiguous identification of SARS-CoV-2 virions.

Immunohistochemistry is a common supplementary method in pathology for diagnostic purposes, but all steps in this technique must be optimised. Antigens are known to be vulnerable at several stages of the immunohistochemical procedure and also during specimen preparation for TEM [9,10]. By carefully selecting protocols for the IHC and TEM procedure and performing all procedures precisely, we preserved good accessibility of the epitopes and also achieved a highly specific reaction on an electron microscopy level.

From our point of view, immunoelectron microscopy is the most reliable method for distinguishing intracellular viral particles from normal cell structures of similar morphology and size as virions. Unfortunately, special and expensive equipment is needed for this methodology. Therefore, we developed a variant of correlative microscopy that allows every routine pathology laboratory to check its results of IHC performed on routinely used paraffin-embedded samples, as well as with a transmission electron microscope without highly developed instruments and sophisticated techniques. Namely, an IHC reaction at a light microscopy level per se could be misleading due to various factors in the pre-analytical phase and misinterpretation of nonspecific staining. Additionally, low magnification and resolution of IHC do not allow pathologists to see virions or be convinced that an IHC reaction colocalizes with virions. Likewise, conventional TEM analysis per se is also not enough for undoubted proof of present virions. Namely, despite the possibility to see the structures (virions) with TEM due to its high resolution, we can misinterpret them. Therefore, only the combination of the localisation of a positive immunoreaction on the
structure of interest (i.e., viruses) and simultaneous identification of this structure on an ultrastructural level gives indisputable proof of the presence of virions. We could therefore recommend pathologists in routine pathology laboratories to confirm their results of IHC against the SARS-CoV-2 virus with correlative microscopy described in our manuscript. In our opinion, this is the main scientific output of our work that can be transferred into everyday practice. We are aware that it represents a small piece of the SARS-CoV-2 jigsaw; however, every piece is important in making the whole picture clearer. We cannot afford false positive or false negative pathology reports these days, especially if we have appropriate knowledge and methodology.

In order to avoid pitfalls in immunogold labelling, appropriate fixation and embedding mediums, which can restrict both antibody access and antibody quality, are crucial [11,12]. In our study, the results of immunogold labelling of SARS-CoV-2 virions strongly depended on the selection of the primary antibody and resin. Only on Lowicryl sections and with an appropriate antibody were we able to get specific staining and a satisfactory ultrastructure. Immunogold labelling of coronaviruses has been reported [13], but we believe this to be the first report to date on the identification of SARS-CoV-2 virions with immunoelectron microscopy.

5. Conclusions

To conclude, immunoelectron microscopy, if performed correctly, is the most reliable method for distinguishing intracellular viral particles from normal cell structures of similar morphology and size as virions. Furthermore, we developed a variant of correlative microscopy that allows pathologists to check the results of IHC performed first on routinely used paraffin-embedded samples, followed by semithin, and finally by ultrathin sections without highly developed instruments and sophisticated techniques. Both methods proved that only the colocalization of a positive immunoreaction on the structure of interest (i.e., viruses) and identification of this structure on an ultrastructural level gives indisputable proof of the presence of virions.”

https://www.ncbi.nlm.nih.gov/labs/pmc/articles/PMC8473209/

In Summary:

• Dr. Harold Hillman asked the question ‘How much does a picture taken with this instrument tell one about the structure of the living cell?’
• TEM permits much higher resolution and magnification than the light microscope, but the tissue can not survive the low pressure, the bombardment of electrons and x-radiation in the electron microscope
• Thus, the tissue has to be coated with a deposit of salts of osmium lead or tungsten, which is not destroyed by these agents, and can therefore be examined
• When one looks at an illustration of an electron micrograph:
  1. An animal has been killed
  2. It cools down
  3. Its tissue is excised
  4. The tissue is fixed (killed)
  5. It is stained with a heavy metal salt
  6. It is dehydrated with increasing concentrations of alcohol
  7. It shrinks
  8. The alcohol is extracted with a fat solvent, propylene oxide
  9. The latter is replaced by an epoxy resin
  10. It hardens in a few days
  11. Sections one tenth of a millimetre thick, or less, are cut
  12. They are placed in the electron microscope, nearly all the air of which is pumped out
  13. A beam of electrons at 10,000 volts to 3,000,000 volts is directed at it
  14. Some electrons strike a phosphorescent screen
  15. The electron microscopists select the field and the magnification which show the features they wish to demonstrate
  16. The image may be enhanced
  17. Photographs are taken and some are selected as evidence
• One can immediately see how far the tissue has travelled from life to an illustration in a book
• Most of the new structures in cells apparent by electron microscopy are artifacts

• Many studies reported detection of supposed “viral” particles by transmission electron microscopy (TEM) whuch was used as sufficient evidence for “viral” invasion of renal tissue but data regarding detection of “viral” RNA or other valuable methods for “viral” detection of kidney specimens were missing
• In one study, the presented particles in the aforementioned studies exhibit a diameter of 50 to 150 nm and crown-like electron-dense coat, so they may appear similar to “coronavirus” but also similar to ubiquitous coated vesicles, such as clathrin-coated vesicles, or COPI- or COPII-coated vesicles
• Moreover, clusters of “viral” particles inside the vacuole might resemble multivesicular bodies (MVBs), which are regular structures of the endocytic pathway
• In a study of 4 patients confirmed with “SARS-COV-2,” “viral” RNA was confirmed in all lung samples, but was negative in all kidney samples
• However, ultrastructural examination revealed intracellular vesicular structures of similar size and morphology in lung with proven “viral” RNA and in kidney with no “viral” RNA
• In lung specimens with proven “viral” RNA, the authors observed many structures that could be either “viral” particles with typical corona or coated vesicles with electron-dense protein coat
• In addition, in the same “SARS-CoV-2–positive” lung specimens, they found vacuoles that could be either membrane-bound clusters of “viral” particles or MVBs with intralumenal vesicles inside
• On the other hand, ultrastructural examination of the lung specimens of 2 patients without “SARS-CoV-2” (1 autopsy specimen of lung with negative RT-PCR for “viral” RNA and 1 biopsy specimen of lung before “COVID” era) revealed the same structures, resembling “viral” particles, coated vesicles, or MVBs, as in a specimen with positive “SARS-CoV-2”
• TEM analysis of postmortem kidney specimens of patients with “COVID-19” revealed numerous individual vesicles and clusters of vesicles in different types of kidney cells, despite negative RT-PCR for “SARS-CoV-2” RNA
• They additionally performed semiquantitative analysis of these intracellular structures in different types of renal cells from 20 preimplantation donor kidney biopsies, before and during the outbreak of “SARS-CoV-2,” with negative RT-PCR for “SARS-CoV-2” RNA to demonstrate that these cell structures are numerous and ubiquitous in various types of cells
• There has been increasing evidence to indicate that coated vesicles and MVBs may mimic “viral” particles
• it is known that the budding of enveloped “viruses” (to which “SARS-CoV-2” belongs) from the plasma membrane, or the limiting membrane of the endosome, resembles the formation of intralumenal vesicles inside MVBs
• In addition, coated vesicles might also faintly resemble “viral” particles
• The authors conclude that although TEM may serve as a useful diagnostic method for the detection of “viral” infection, caution should be exercised when confirmation of “viral” invasion relies only on TEM
• Additional convincing methods, including immunoelectron microscopy, immunohistochemistry, and “viral” genetic material analysis, are needed for indisputable proof of “viral” invasion in organs

• There is increasing evidence that identification of “SARS-CoV-2 virions” by transmission electron microscopy could be misleading due to the similar morphology of “virions” and ubiquitous cell structures
• According to the authors, immunoelectron microscopy is the most reliable method for distinguishing intracellular “viral” particles from normal cell structures of similar morphology and size as “virions”
• Few researchers have drawn attention to the possible misinterpretation of TEM findings of “SARS-COV-2” due to the similarity of structure and size between “virions” and normal cell structures
• In a previous study, the authors state that they showed cell structures resembling “SARS-CoV-2 virions” in lung and kidney specimens of “SARS-CoV-2” positive and negative patients based on their RT-PCR results for “SARS-CoV-2” RNA
• Existing methods used for “viral” detection in tissue specimens have known limitations
• Immunohistochemistry can be falsely positive or negative due to several reasons in the pre-analytical or analytical phase
• Conventional TEM analysis strongly depends on tissue preservation, which might distort the structure of “viruses” if it is not performed optimally
• An additional disadvantage of TEM analysis is that the sample might miss the area containing “viruses”
• The authors performed a variant of correlative immunohistochemistry and immunogold labelling of “SARS-CoV-2” proteins for indisputable proof of the presence of “SARS-CoV-2 virions” in nasopharyngeal tissue of patients with positive RT-PCR for “SARS-CoV-2” RNA (note they did not mention any controls using NP tissue from “SARS-COV-2” negative patients)
• They claimed to show that a specific and reliable immunoreaction crucially depends on the immunolabelling protocol, from the first steps of tissue sample preparation to the choice of antibody
• I’ve included the outline for each process the samples are put through so that you can see how many substances they are subjected to and how many alterations they undergo
• The Immunohistochemistry Process:
  1. Nasopharyngeal tissue pieces were fixed in 4% paraformaldehyde for 24 h, dehydrated in ethanol, and embedded in paraffin
  2. The tissue sample for immunohistochemistry was obtained by punching the paraffin block with a manual tissue punch
  3. Punched paraffin blocks were then deparaffinized and rehydrated with ethanol
  4. Antigen retrieval was performed with microwave heating in 10 mM sodium citrate buffer, pH 6.0 for 20 min
  5. Endogenous peroxidases were blocked by 3% hydrogen peroxide for 15 min following incubation in primary rabbit antibodies against “SARS-CoV-2” nucleocapsid protein for 2 h at room temperature
  6. After washing with reaction buffer an UltraView Universal DAB Detection Kit was used with the following protocol:
     • HRP enzyme conjugated to goat anti-rabbit secondary antibodies for 20 min
     • Reaction buffer twice for 5 min
     • A mixture of 3,3’-diaminobenzidine (DAB) chromogen and H2O2 for 20 min
     • Washed with reaction buffer and distilled water for 5 min
• The Transmission Electron Microscope Process:
  1. After IHC, tissue samples were transferred to 0.1 M Millonig’s phosphate buffer overnight
  2. They were first post-fixed in 1% OsO4 for 30 min
  3. They were dehydrated in graded concentrations of ethanol and propylene oxide 1-2-propylene oxide for 10 min in each solution
  4. Tissue pieces were then incubated in a mixture (1:1) of 1-2-propylene oxide and Epon 812 resin for 20 min
  5. They were embedded in 100% Epon
  6. Then polymerized at 60 ◦C for 24 h
  7. The next day, semithin sections of 1 µm were cut with an ultramicrotome Leica EM UC6 and stained with Azure II staining solution to find “SARS-CoV-2-positive” cells as the region of interest, to select this region for ultrathin sectioning
  8. Ultrathin sections of 60 nm were cut and viewed in a transmission electron microscope
• The Immunogold Labelling on Ultrathin Sections Process (Epon sections):
  1. Small samples (2 mm3) of nasopharyngeal tissue were fixed in a mixture of 4% paraformaldehyde and 2% glutaraldehyde in 0.2 M cacodylate buffer (pH 7.3) for 3 h at 4 ◦C
  2. Overnight rinsing in 0.33 M sucrose in 0.2 M cacodylate buffer was followed by post-fixation with 1% OsO4 for 1 h
  3. After dehydration in an ethanol series, tissue samples were embedded in Epon 812 resin and cut into ultrathin sections (60 nm thick), which were collected on nickel grids
  4. Antigen unmasking treatment was performed by incubation of some grids in a humid chamber on a large drop of a saturated aqueous solution of sodium meta periodate for 1 h at room temperature
  5. Grids were then washed in distilled water
  6. Non-specific labelling was blocked by blocking buffer (5% foetal calf serum in 0.1% BSA in PBS (washing buffer)) for 30 min at room temperature
  7. Primary antibodies against “SARS-CoV-2” nucleocapsid protein or “SARS-CoV-2” spike glycoprotein S1 diluted 1:100 was applied and incubated overnight at 4 ◦C
  8. After washing in washing buffer, goat anti-rabbit secondary antibodies with 18 nm gold (Au) diluted 1:40 in blocking buffer were applied for 90 min
  9. In all cases, negative controls were also performed, in which primary antibodies were replaced with PBS
  10. Sections were counterstained with uranyl acetate and lead citrate
• The Immunogold Labelling on Ultrathin Sections Process (Lowicryl sections):
  1. Small cubes (1 mm 3) of nasopharyngeal tissue were transferred to 2% paraformaldehyde plus 0.05% glutaraldehyde in PBS for 1 h at room temperature
  2. Samples were dehydrated by progressively lowering the temperature
  3. Embedded in Lowicryl HM20 resin in a Leica AFS apparatus according to the following protocol:
     • 30% ethanol for 30 min at 0 ◦C
     • 55% ethanol for 30 min at −15 ◦C
     • 70% ethanol for 30 min at −30 ◦C
     • 100% ethanol for 1 h at −50 ◦C
     • 75% ethanol/25% HM20 for 1 h at −50 ◦C
     • 50% ethanol/50% HM20 for 1 h at −50 ◦C
     • 25% ethanol/75% HM20 for 1 h at −50 ◦C
     • 100% HM20 for 1 h, and 100% HM20 overnight at −50 ◦C
  4. HM20 was polymerized for 48 h at −50 ◦C and then for 24 h at −20 ◦C under UV light
  5. Ultrathin sections (60 nm thick) were cut and collected on nickel grids
  6. Sections were washed in washing buffer (0.1% Na-azide, 0.8% BSA, and 0.1% IGSS gelatine in PBS)
  7. Blocked in blocking buffer (5% foetal calf serum in 0.1% BSA in PBS (washing buffer)) for 30 min at room temperature
  8. Incubated overnight at 4 ◦C with primary antibodies against “SARS-CoV-2” nucleocapsid protein diluted 1:50 or “SARS-CoV-2” spike glycoprotein S1 diluted 1:100.
  9. After washing in washing buffer, goat anti-rabbit secondary antibodies with 18 nm gold (Au) diluted 1:40 in blocking buffer were applied for 90 min
  10. Sections were counterstained with uranyl acetate and lead citrate
• The authors state that through immunogold labelling, they confirmed the presence of “SARS-CoV-2 virions” in human nasopharyngeal tissue samples
• However, the results of this method greatly depended on the type of ultrathin sections and primary antibodies used in the immunolabelling protocol
• Immunogold reaction on ultrathin Epon sections with primary antibodies against “SARS-CoV-2” nucleocapsid protein was ubiquitous and therefore unspecific
• On ultrathin Epon sections with primary antibodies against “SARS-CoV-2” spike glycoprotein S1, it was scarce and unspecific
• After an antigen unmasking procedure on ultrathin Epon sections with primary antibodies against “SARS-CoV-2” spike glycoprotein S1, immunogold labelling was more “specific” but weak
• It wasn’t until they performed immunogold labelling on ultrathin Lowicryl sections with primary antibodies against “SARS-CoV-2” spike glycoprotein S1 that their reaction was “specific” and intense
• “Viral” genetic material analysis of patients’ nasopharyngeal tissue by itself is not sufficient proof of “SARS-CoV-2” active infection in body tissues, but a positive RT-PCR test is a precondition for further analysis of “SARS-CoV-2 virus” presence in a particular tissue
• TEM analysis per se is not enough for unequivocal proof of “SARS-CoV-2 virions” in the tissue of interest due to the similar structure and size of these “virions” and ubiquitous cell structures
• As these steps alone are not valid proof, the authors performed correlative microscopy in which they combined the advantages of both IHC and electron microscopy to analyse the same specimen
• Immunohistochemistry is a common supplementary method in pathology for diagnostic purposes, but all steps in this technique must be optimised
• Antigens are known to be vulnerable at several stages of the immunohistochemical procedure and also during specimen preparation for TEM
• From their point of view, immunoelectron microscopy is the most reliable method for distinguishing intracellular “viral” particles from normal cell structures of similar morphology and size as “virions”
• However, the authors admit that a IHC reaction at a light microscopy level per se could be misleading due to various factors in the pre-analytical phase and misinterpretation of nonspecific staining
• Additionally, low magnification and resolution of IHC do not allow pathologists to see “virions” or be convinced that an IHC reaction colocalizes with “virions”
• Likewise, conventional TEM analysis per se is also not enough for undoubted proof of present “virions”
• Namely, despite the possibility to see the structures (“virions”) with TEM due to its high resolution, they can be misinterpreted
• Only the combination of the localisation of a positive immunoreaction on the structure of interest (i.e., “viruses”) and simultaneous identification of this structure on an ultrastructural level gives indisputable proof of the presence of “virions”
• In other words, the authors believe that combining two techniques which they admit by themselves can not be considered proof is somehow considered proof
• In order to avoid pitfalls in immunogold labelling, appropriate fixation and embedding mediums, which can restrict both antibody access and antibody quality, are crucial
• In their study, the results of immunogold labelling of “SARS-CoV-2 virions” strongly depended on the selection of the primary antibody and resin
• Only on Lowicryl sections and with an appropriate antibody were they able to get “specific” staining and a satisfactory ultrastructure
• The authors conclude that immunoelectron microscopy, if performed correctly, is the most reliable method for distinguishing intracellular “viral” particles from normal cell structures of similar morphology and size as “virions”

What exactly are the particles that were selected from a sea of billions of lookalikes to be used as the representation of “SARS-COV-2?” Are they coated vesicles, multivesicular bodies, exosomes, golgi and costomer-coated vesicles, secretory vesicles, and/or clathrin-coated vesicles? Are they all or none of the above? Are they simply artefacts created through the multiple structural-altering processes the samples are put through in order to obtain the images as Dr. Harold Hillman showed with his underappreciated research?

Whatever they are, it is absolutely impossible to claim that these particles are “SARS-COV-2” or any other “virus” as they were never properly purified nor isolated directly from the samples of sick humans. This is the very reason the researchers can not distinguish the particles they believe to be “SARS-COV-2” from any of the other identical or nearly identical normal “non-viral” particles within the sample. They have never had the exact particles they claim as “SARS-COV-2” separated and freed from everything else (i.e. isolated). Absolute isolation is the only way that any researcher could be entirely certain that the particles they are working with are the ones they believe to be the cause of disease. It is the only way that they can prove pathogeniticity by adhering to the scientific method. It is the only way they could biochemically and molecularlly characterize the particles. It is the only way that they could attempt to show any specificity with certain antibodies. It is the only way that the TEM images would be of any value whatsoever as proof of “viral” particles.

Without purifying and isolating the particles believed to be “SARS-COV-2,” we get confusing, false, and erroneous results. We get one set of researchers claiming the “corona” particles as a novel “virus” while another set of researchers claim they are multivesicular bodies and/or other normal subcellular particles. We get non-specific antibody results claimed to be specific yet the specificity is unable to be shown and reproduced by independent researchers. We get “SARS-COV-2” particles found in samples with confirmed “viral” RNA as well as in those without any “viral” RNA whatsoever. Most importantly, we get “SARS-COV-2” particles found in the samples from PCR’-negative patients taken before the “virus” was said to even exist.

Putting it all together, it is abundantly clear that the images of the particles claimed to be “SARS-COV-2” are as meaningless as the results of the “viral” genetic material obtained by way of PCR. They are as meaningless as the “specific” antibodies that regularly bind to non-target material. They are as meaningless as the computer-generated stitched together genomes taken from unpurified sources. In every measurable way possible, the indirect results said to identify “viral” entities never shown to exist in a purified and isolated state are utterly meaningless. These enhanced images of non-specific particles only hold value to those who have manipulated you through fear and propaganda. Do not let them hold any more power over you going forward.

When they can find the exact same “viral” particles in those said to not have the “virus,” it is time for virology to admit the obvious: